Expeditionary Mechanical Ventilation in Conjunction With Extracorporeal Life Support During Ground Transport.

BACKGROUND: We assessed the use of an FDA-cleared transport ventilator with limited functions and settings during ground transport in a swine model of ground evacuation. We hypothesized that when used as an adjunct to extracorporeal life support (ECLS), the device would enable safe mobile ventilatory support during ground evacuation. METHODS: Female Yorkshire swine (n = 11; mean, 52.4 ± 1.3 kg) were sedated and anesthetized and received mechanical ventilation (MV) with a standard intensive care unit (ICU) ventilator and were transitioned to the Simplified Automated Ventilator II (SAVe II; AutoMedx) during ground transport. MV served as an adjunct to ECLS in all animals. Ventilator performance was assessed in the uninjured state on day 1 and after bilateral pulmonary contusion on day 2. Data were collected pre- and post-transport on both days. RESULTS: During 33 transports, the SAVe II provided similar ventilation support as the ICU ventilator. Mean total transport time was 38.8 ± 2.1 minutes. The peak inspiratory pressure (PIP) limit was the only variable to show consistent differences pre- and post-transport and between ventilators. No adverse events occurred. CONCLUSION: As an adjunctive supportive device during ground transport, the SAVe II performed adequately without failure or degradation in subject status. Further testing is warranted to elucidate the clinical limits of this device during standalone use.

Dynamics of appearance and decay of gaseous microemboli during in vitro extracorporeal circulation.

INTRODUCTION: Extracorporeal life support (ECLS) patients are at risk for complications caused by gaseous microemboli (GME). GMEs can cause hypoxia, inflammation, coagulation, and end-organ damage. The objective of this in vitro study was to assess dynamics of GME formation during circulation of whole blood or a glycerol blood surrogate. We hypothesized that there is no difference in GME counts and sizes between whole blood and the glycerol blood surrogate and that the membrane lung reduces GME counts over time. METHODS: A circulation platform was developed using the Cardiohelp ECLS system to run either donor blood or glycerol solution. We conducted 10 repetitions consisting of three phases of ultrasound GME detection using the EDAC™ Quantifier (Luna Innovations, Charlottesville, VA, USA) for each group. Phases were 3-minute recordings at the initiation of 2 L/min flow (Phase 1), post-injection of a GME suspension (Phase 2), and 10 minutes after injection (Phase 3). The number and size of GME pre- and post-ML were recorded separately and binned based on diameter ranges. RESULTS: In Phase 1, GME count in blood was higher than in glycerol. In Phase 2, there was a large increase in GME counts; however, most GME were reduced post-membrane in both groups. In Phase 3, there was a significant decrease in GME counts compared to Phase 2. GME > 100 µm in glycerol decreased post membrane. CONCLUSIONS: We demonstrated GME formation and decay dynamics during in vitro circulation in an ECLS system with blood and glycerol. GME counts were higher in blood, likely due to varying rheological properties. There were decreases in GME levels post membrane in both groups after GME injection, with the membrane lung effectively trapping the GME, and additional reduction 10 minutes after GME injection.

Tethered Liquid Perfluorocarbon Coating for 72 Hour Heparin-Free Extracorporeal Life Support.

Coagulopathic complications during extracorporeal life support (ECLS) result from two parallel processes: 1) foreign surface contact and shear stress during blood circulation and 2) administration of anticoagulant drugs to prevent circuit thrombosis. To address these problems, biocompatible surfaces are developed to prevent foreign surface-induced coagulopathy, reducing or eliminating the need for anticoagulants. Tethered liquid perfluorocarbon (TLP) is a nonadhesive coating that prevents adsorption of plasma proteins and thrombus deposition. We examined application of TLP to complete ECLS circuits (membranes, tubing, pumps, and catheters) during 72 hours of ECLS in healthy swine (n = 5/group). We compared TLP-coated circuits used without systemic anticoagulation to standard of care: heparin-coated circuits with continuous heparin infusion. Coagulopathic complications, device performance, and systemic effects were assessed. We hypothesized that TLP reduces circuit thrombosis and iatrogenic bleeding, without impeding gas exchange performance or causing untoward effects. No difference in bleeding or thrombotic complication rate was observed; however, circuit occlusion occurred in both groups (TLP = 2/5; CTRL = 1/5). TLP required elevated sweep gas rate to maintain normocapnia during ECLS versus CTRL (10-20 vs. 5 L/min; p = 0.047), suggesting impaired gas exchange. Thrombus deposition and protein adhesion on explanted membranes were comparable, and TLP did not preserve platelet or blood cell counts relative to controls. We conclude that neither TLP nor standard of care is an efficacious solution to prevent coagulation disturbances during ECLS. Further testing of promising biomaterials for ECLS utilizing the model outlined here is warranted.

Noninvasive SpO2/FiO2 ratio as surrogate for PaO2/FiO2 ratio during simulated prolonged field care and ground and high-altitude evacuation.

BACKGROUND: Diagnosis of lung injury requires invasive blood draws to measure oxygen tension in blood. This capability is nonexistent in austere settings and during prolonged field care (PFC), that is, medical care characterized by inability to evacuate casualties from the point of injury for up to 72 hours. We analyzed pulse-oximeter-derived noninvasive SpO2 and assessed the SpO2/FiO2 ratio (SFR) as a surrogate for the PaO2/FiO2 ratio (PFR), an accepted marker of lung function. We hypothesized that SFR is a suitable surrogate for PFR in a data set from animal models of combat-relevant trauma, PFC, and aeromedical evacuation. METHODS: Data from anesthetized swine (N = 30) subjected to combat relevant trauma, resuscitation, and critical care interventions were analyzed. Pairwise correlations and Bland-Altman and regression analyses were performed to compare PFR and SFR, based on averaged SpO2 values obtained from two monitoring devices. RESULTS: We performed 683 pairwise correlations. SpO2/FiO2 ratio was numerically higher than PFR with a 313 cutoff values for acute respiratory distress syndrome (ARDS) (PFR ≥300). Sensitivity/specificity for detection of mild ARDS was 75%/73% with a 200 to 300 PFR range corresponding to 252 to 312 SFR range. For moderate ARDS, sensitivity/specificity was 61%/93% with a 100 to 200 PFR range corresponding to 191 to 251 SFR range. For severe ARDS, sensitivity/specificity was 49%/97% with a 0 to 100 PFR range corresponding to 0 to 190 SFR range. For all groups, areas under the receiver operating characteristic curves ranged from 0.76 to 0.98. CONCLUSION: SpO2/FiO2 ratio is a useful surrogate for PFR when arterial blood gas testing is not available during dynamically changing physiologic conditions, for example, during austere conditions, PFC, or aeromedical evacuation, and may permit early detection of casualties in need of lung-specific life-saving interventions. Studies in critically ill humans are warranted.

Expression of High Mobility Group Box 1 Protein in a Polytrauma Model During Ground Transport and Simulated High-Altitude Evacuation.

BACKGROUND: We investigated the expression of high mobility group box 1 (HMGB1) protein in a combat-relevant polytrauma/ acute respiratory distress syndrome (ARDS) model. We hypothesized that systemic HMGB1 expression is increased after injury and during aeromedical evacuation (AE) at altitude. METHODS: Female Yorkshire swine (n =15) were anesthetized and cannulated with a 23Fr dual-lumen catheter. Venovenous extracorporeal life support (VV ECLS) was initiated via the right jugular vein and carried out with animals uninjured on day 1 and injured by bilateral pulmonary contusion on day 2. On both days, animals underwent transport and simulated AE. Systemic HMGB1 expression was measured in plasma by ELISA. Plasma-free Hb (pfHb) was measured with the use of spectrophotometric methods. RESULTS: Plasma HMGB1 on day 1 was transiently higher at arrival to the AE chambers, increased significantly after injury, reaching highest values at 8,000 ft on day 2, after which levels decreased but remained elevated versus baseline at each time point. pfHb decreased on day 1 at 30,000 ft and significantly increased on day 2 at 8,000 ft and postflight. CONCLUSIONS: Systemic HMGB1 demonstrated sustained elevation after trauma and altitude transport and may provide a useful monitoring capability during en route care.

Heparin-Free Extracorporeal Life Support Using Tethered Liquid Perfluorocarbon: A Feasibility and Efficacy Study.

Coagulation management is the leading challenge during extracorporeal life support (ECLS) due to shear stress and foreign-surface-induced coagulation disturbance during circulation. A nonadhesive, liquid-infused coating called tethered liquid perfluorocarbon (TLP) was developed to prevent adhesion of blood on medical materials. We investigated the novel application of TLP to commercial ECLS circuits compared with standard heparin-coated circuits in vivo in anesthetized swine for 6 hours veno-venous ECLS (1 L/min blood flow) without systemic anticoagulation (n = 3/group). We hypothesized that TLP coating permits heparin-free circulation without untoward effects while reducing thrombus deposition compared with controls. Vital signs, respiration, gas transfer, coagulation, and histology were assessed. Scanning electron microscopy (SEM), elemental mapping, and digital imaging were used to assess thrombus deposition after circulation. There were no group differences in vitals, gas exchange, coagulation, and histology. In both groups, ECLS enabled a decrease in minute volume and end-tidal CO2, with concomitant increase in pH (p < 0.05). Scanning electron microscopy and digital imaging revealed significant thrombus on heparin-coated membranes, which was reduced or absent on TLP-coated materials. Tethered liquid perfluorocarbon permitted heparin-free ECLS without altering device performance and prevented thrombus deposition versus immobilized heparin. Pending multiday in vivo testing, TLP is a promising biomaterial solution to eliminate anticoagulation requirements during ECLS.

Effects of adjunct treatments on end-organ damage and histological injury severity in acute respiratory distress syndrome and multiorgan failure caused by smoke inhalation injury and burns.

BACKGROUND: We investigated effects of mesenchymal stem cells (MSC) or low-flow extracorporeal life support (ECLS) as adjunctive treatments for acute respiratory distress syndrome (ARDS) due to inhalation injury and burns. We hypothesized that these interventions decrease histological end-organ damage. METHODS: Anesthetized female swine underwent smoke inhalation injury and 40% TBSA burns, then critical care for 72h. The following groups were studied: CTR (no injury, n = 4), ICTR (injured untreated, n = 10), Allo (injured treated with allogenic MSC, n = 10), Auto (injured treated with autologous MSC, n = 10), Hemo (injured and treated with the Hemolung low flow ECLS system, n = 9), and Nova (injured and treated with the NovaLung low flow ECLS system, n = 8). Histology scores from lung, kidneys, liver, and jejunum were calculated. Data are presented as means±SEM. RESULTS: Survival at 72h was 100% in CTR; 40% in ICTR; 50% in Allo; 90% in Auto; 33% in Hemo; 63% in Nova. ARDS developed in 0/10 CTR; 10/10 ICTR; 8/9 Hemo; 5/8 Nova; 9/10 Allo; 6/10 Auto. Diffuse alveolar damage (DAD) was present in all injured groups. MSC groups had significantly lower DAD scores than ICTR animals (Allo 26.6 ± 3.4 and Auto 18.9 ± 1.5 vs. ICTR 46.8 ± 2.1, p < 0.001). MSC groups also had lower DAD scores than ECLS animals (Allo vs. Nova, p < 0.05, Allo vs. Hemo p < 0.001, Auto vs. Nova p < 0.001, Auto vs. Hemo, p < 0.001). Kidney injury ICTR (p < 0.05) and Hemo (p < 0.01) were higher than in CTR. By logistic regression, a PaO2-to-FiO2 ratio (PFR) < 300 was a function of the DAD score: logit (PFR < 300) = 0.84 + 0.072*DAD Score, odds ratio 1.074 (1.007, 1.147, p < 0.05) with a ROC AUC of 0.76, p < 0.001. CONCLUSION: Treatment with Auto MSC followed by Allo and then Nova were most effective in mitigating ARDS and MOF severity in this model. Further studies will elucidate the role of combination therapies of MSC and ECLS as comprehensive treatments for ARDS and MOF.

Altered expression of aquaporin 1 and aquaporin 5 in the cornea after primary blast exposure.

Purpose: Our study aimed to determine whether the altered expression of biomarkers linked to corneal injuries, such as the edema-regulating proteins aquaporin-1 and aquaporin-5 (AQP1 and AQP5), occurred following primary blast exposure. Methods: Adult male Dutch Belted rabbits were anesthetized and exposed to blast waves with peak overpressures of 142.5-164.1 kPa (20.4-23.4 psi). These exposure groups experienced peak blast overpressure-specific impulses (impulse per unit surface area) of 199.6-228.5 kPa-ms. Unexposed rabbits were included as controls. The animals were euthanized at 48 h post-exposure. Corneas obtained from the euthanized blast-exposed and control rabbits were processed for quantitative PCR and western blot to quantify mRNA and the protein expression of AQP1 and AQP5. Immunohistochemical analysis was conducted to determine the cellular localization of AQP1 and AQP5. Results: Corneal thickness increased up to 18% with the peak blast overpressure-specific impulses of 199.6-228.5 kPa-ms at 48 h after blast exposure. mRNA levels of AQP1 and AQP5 increased in the whole cornea lysates of blast-exposed rabbits relative to those of the controls. Western blot analyses of whole cornea lysates revealed that the expression levels of AQP1 and AQP5 were approximately 2- and 1.5-fold higher, respectively, in blast-exposed rabbits compared to controls. The extent of AQP1 immunostaining (AQP1-IS) increased in the epithelial cell layer after blast exposure. The AQP5-IS pattern changed from a mixed membrane and cytoplasmic expression in the controls to predominantly cytoplasmic expression in the basally located cornea epithelial cells of blast-exposed rabbits. Conclusions: Primary blast exposure resulted in edema-related changes in the cornea manifested by the altered expression of the edema-regulating proteins AQP1 and AQP5 with blast overpressure-specific impulses. These findings support potential acute corneal injury mechanisms in which the altered regulation of water permeability is caused by primary blast exposure.

Dynamics of acute respiratory distress syndrome development due to smoke inhalation injury: Implications for prolonged field care.

BACKGROUND: Smoke inhalation injury (SII) causes 30% to 40% mortality and will increase as a cause of death during prolonged field care. We used a combat relevant model of acute respiratory distress syndrome due to SII to study temporal changes in ventilation-perfusion (V/Q) matching, computed tomography (CT) scan data, and histopathology and hypothesized that SII leads to increase in shunt (Qshunt), V/Q mismatch, lung consolidation, and diffuse alveolar damage. METHODS: Swine received severe SII and airway pressure release ventilation (APRV, n = 6), or conventional ARDSNet mechanical ventilation (MV) (CMV, n = 8). A control group without injury received volume controlled MV (CTRL, n = 6), The multiple inert gas elimination technique and CT were performed at baseline (BL), 0.5 hours, 1 hours, 2 hours, 24 hours, and 48 hours after injury. Diffuse alveolar damage scoring was performed post mortem. Significance at p less than 0.05: APRV versus CTRL; CMV versus CTRL; APRV versus CMV*; denotes changes versus BL. RESULTS: (1) SII caused increases in Qshunt more so in APRV than CMV group. Qshunt did not change in CTRL. (2) PaO2-to-FIO2 ratio (PFR) was lower in APRV versus CTRL at 2 hours (375 ± 62‡ vs. 549 ± 40) and 24 hours (126 ± 34‡* vs. 445 ± 5) and 48 hours (120 ± 41‡& vs. 430 ± 13). In CMV animals, PFR was lower versus CTRL and BL at 24 hours (238 ± 33) and 48 hours (98 ± 27). Qshunt correlated with PFR (r = 0.75, p < 0.0001, APRV and (r = 0.65, p < 0.0001, CMV). CT showed decrease in normally aerated lung, while poorly and nonaerated lung increased. CONCLUSION: Smoke inhalation injury leads to early development of shunt, V/Q mismatch, lung consolidation, and diffuse alveolar damage. These data substantiate the need for new point of injury interventions in the prolonged field care setting. LEVEL OF EVIDENCE: Animal research.

Thromboelastography on-the-go: Evaluation of the TEG 6s device during ground and high-altitude Aeromedical Evacuation with extracorporeal life support.

BACKGROUND: Coagulation monitoring capabilities during transport are limited. Thromboelastography (TEG) is a whole-blood clotting test measuring clot formation, stabilization, and fibrinolysis and is traditionally performed in a laboratory. We evaluated a new point-of-care TEG analyzer, TEG 6s (Haemonetics, Braintree, MA), in a large animal model of combat-relevant trauma managed with extracorporeal life support during ground and high-altitude aeromedical evacuation. The objective was to compare TEG 6s used during transport versus the predicate device, TEG 5000, used in the laboratory. We hypothesized that TEG 6s would be comparable with TEG 5000 during dynamically changing transport conditions. METHODS: Thromboelastography parameters (R, K, angle, MA, LY30) derived by TEG 6s and TEG 5000 were compared during transport of 8 swine. TEG 6s was transported with animals during ground transport and flight. TEG 5000 was stationary in an adjacent building. TEG 6s activated clotting time (ACT) was compared with a Hemochron Junior ACT analyzer (Accriva Diagnostics, San Diego, CA). Statistics were performed using SAS 9.4 with Deming regressions, Spearman correlations, and average differences compared. RESULTS: Correlation between devices was stronger at sea-level (R, r = 0.7413; K, r = 0.7115; angle, r = 0.7192; MA, r = 0.8386; LY30, r = 0.9099) than during high-altitude transport (R, r = 0.4787; K, r = 0.4007; angle, r = 0.3706; MA, r = 0.6573; LY30, r = 0.8481). Method agreement was comparable during stationary operation (R, r = 0.7978; K, r = 0.7974; angle, r = 0.7574; MA, r = 0.7841; LY30, r = 0.9140) versus ground transport (R, r = 0.7927; K, r = 0.6246; angle, r = 0.6967; MA, r = 0.9163; LY30, r = 0.8603). TEG 6s ACT trended higher than Hemochron ACT when subjects were heparinized (average difference, 1,442 ± 1,703 seconds) without a methodological difference by Deming regression. CONCLUSION: Mobile TEG 6s during ground and altitude transport is feasible and provides unprecedented information to guide coagulation management. Future studies should assess the precision and accuracy of TEG 6s during transport of critically ill.

Automatic proximal airway volume segmentation using optical coherence tomography for assessment of inhalation injury.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury with a mortality rate of up to 40%. Early management of ARDS has been difficult due to the lack of sensitive imaging tools and robust analysis software. We previously designed an optical coherence tomography (OCT) system to evaluate mucosa thickness (MT) after smoke inhalation, but the analysis relied on manual segmentation. The aim of this study is to assess in vivo proximal airway volume (PAV) after inhalation injury using automated OCT segmentation and correlate the PAV to lung function for rapid indication of ARDS. METHODS: Anesthetized female Yorkshire pigs (n = 14) received smoke inhalation injury (SII) and 40% total body surface area thermal burns. Measurements of PaO2-to-FiO2 ratio (PFR), peak inspiratory pressure (PIP), dynamic compliance, airway resistance, and OCT bronchoscopy were performed at baseline, postinjury, 24 hours, 48 hours, 72 hours after injury. A tissue segmentation algorithm based on graph theory was used to reconstruct a three-dimensional (3D) model of lower respiratory tract and estimate PAV. Proximal airway volume was correlated with PFR, PIP, compliance, resistance, and MT measurement using a linear regression model. RESULTS: Proximal airway volume decreased after the SII: the group mean of proximal airway volume at baseline, postinjury, 24 hours, 48 hours, 72 hours were 20.86 cm (±1.39 cm), 17.61 cm (±0.99 cm), 14.83 cm (±1.20 cm), 14.88 cm (±1.21 cm), and 13.11 cm (±1.59 cm), respectively. The decrease in the PAV was more prominent in the animals that developed ARDS after 24 hours after the injury. PAV was significantly correlated with PIP (r = 0.48, p < 0.001), compliance (r = 0.55, p < 0.001), resistance (r = 0.35, p < 0.01), MT (r = 0.60, p < 0.001), and PFR (r = 0.34, p < 0.01). CONCLUSION: Optical coherence tomography is a useful tool to quantify changes in MT and PAV after SII and burns, which can be used as predictors of developing ARDS at an early stage. LEVEL OF EVIDENCE: Prognostic, level III.

Point-of-care endoscopic optical coherence tomography detects changes in mucosal thickness in ARDS due to smoke inhalation and burns.

BACKGROUND: The prevalence of acute respiratory distress syndrome (ARDS) in mechanically ventilated burn patients is 33%, with mortality varying from 11-46% depending on ARDS severity. Despite the new Berlin definition for ARDS, prompt bedside diagnosis is lacking. We developed and tested a bedside technique of fiberoptic-bronchoscopy-based optical coherence tomography (OCT) measurement of airway mucosal thickness (MT) for diagnosis of ARDS following smoke inhalation injury (SII) and burns. METHODS: 16 female Yorkshire pigs received SII and 40% thermal burns. OCT MT and PaO2-to-FiO2 ratio (PFR) measurements were taken at baseline, after injury, and at 24, 48, and 72h after injury. RESULTS: Injury led to thickening of MT which was sustained in animals that developed ARDS. Significant correlations were found between MT, PFR, peak inspiratory pressure (PIP), and total infused fluid volume. CONCLUSIONS: OCT is a useful tool to quantify MT changes in the airway following SII and burns. OCT may be effective as a diagnostic tool in the early stages of SII-induced ARDS and should be tested in humans.

Low-Level Blast Exposure Increases Transient Receptor Potential Vanilloid 1 (TRPV1) Expression in the Rat Cornea.

BACKGROUND: Blast-related ocular injuries sustained by military personnel have led to rigorous efforts to elucidate the effects of blast exposure on neurosensory function. Recent studies have provided some insight into cognitive and visual deficits sustained following blast exposure; however, limited data are available on the effects of blast on pain and inflammatory processes. Investigation of these secondary effects of blast exposure is necessary to fully comprehend the complex pathophysiology of blast-related injuries. The overall purpose of this study is to determine the effects of single and repeated blast exposure on pain and inflammatory mediators in ocular tissues. METHODS: A compressed air shock tube was used to deliver a single or repeated blast (68.0 ± 2.7 kPa) to anesthetized rats daily for 5 days. Immunohistochemistry was performed on ocular tissues to determine the expression of the transient receptor potential vanilloid 1 (TRPV1) channel, calcitonin gene-related peptide (CGRP), substance P (SP), and endothelin-1 (ET-1) following single and repeated blast exposure. Neutrophil infiltration and myeloperoxidase (MPO) expression were also assessed in blast tissues via immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) analysis, respectively. RESULTS: TRPV1 expression was increased in rat corneas exposed to both single and repeated blast. Increased secretion of CGRP, SP, and ET-1 was also detected in rat corneas as compared to control. Moreover, repeated blast exposure resulted in neutrophil infiltration in the cornea and stromal layer as compared to control animals. CONCLUSION: Single and repeated blast exposure resulted in increased expression of TRPV1, CGRP, SP, and ET-1 as well as neutrophil infiltration. Collectively, these findings provide novel insight into the activation of pain and inflammation signaling mediators following blast exposure.

Low-Level Primary Blast Causes Acute Ocular Trauma in Rabbits.

The objective of this study was to determine whether clinically significant ocular trauma can be induced by a survivable isolated primary blast using a live animal model. Both eyes of 18 Dutch Belted rabbits were exposed to various survivable low-level blast overpressures in a large-scale shock tube simulating a primary blast similar to an improvised explosive device. Eyes of the blast-exposed rabbits (as well as five control rabbits) were thoroughly examined before and after blast to detect changes. Clinically significant changes in corneal thickness arose immediately after blast and were sustained through 48 h, suggesting possible disruption of endothelial function. Retinal thickness (RT) increased with increasing specific impulse immediately after exposure. Intraocular pressure (IOP) was inversely correlated with the specific impulse of the blast wave. These findings clearly indicate that survivable primary blast causes ocular injuries with likely visual functional sequelae of clinical and military relevance.

Deriving retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells by different sizes of embryoid bodies.

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

Pathophysiology of blast-induced ocular trauma in rats after repeated exposure to low-level blast overpressure.

BACKGROUND: The incidence of blast-induced ocular injury has dramatically increased due to advances in weaponry and military tactics. A single exposure to blast overpressure (BOP) has been shown to cause damage to the eye in animal models; however, on the battlefield, military personnel are exposed to BOP multiple times. The effects of repeated exposures to BOP on ocular tissues have not been investigated. The purpose of this study is to characterize the effects of single or repeated exposure on ocular tissues. METHODS: A compressed air shock tube was used to deliver 70 ± 7 KPa BOP to rats, once (single blast overpressure [SBOP]) or once daily for 5 days (repeated blast overpressure [RBOP]). Immunohistochemistry was performed to characterize the pathophysiology of ocular injuries induced by SBOP and RBOP. Apoptosis was determined by quantification activated caspase 3. Gliosis was examined by detection of glial fibrillary acidic protein (GFAP). Inflammation was examined by detection of CD68. RESULTS: Activated caspase 3 was detected in ocular tissues from all animals subjected to BOP, while those exposed to RBOP had more activated caspase 3 in the optic nerve than those exposed to SBOP. GFAP was detected in the retinas from all animals subjected to BOP. CD68 was detected in optic nerves from all animals exposed to BOP. CONCLUSION: SBOP and RBOP induced retinal damage. RBOP caused more apoptosis in the optic nerve than SBOP, suggesting that RBOP causes more severe optic neuropathy than SBOP. SBOP and RBOP caused gliosis in the retina and increased inflammation in the optic nerve.

Pathophysiology of blast-induced ocular trauma with apoptosis in the retina and optic nerve.

BACKGROUND: Blast-induced ocular trauma is a frequent cause of morbidity for survivors of improvised explosive devices. Blast overpressure (BOP) of 120 ± 7 KPa has been shown to cause damage to lungs, brain, and gut in a rat model; however, the effects of BOP on ocular tissues have not been characterized. To elucidate the pathophysiology of blast-induced ocular trauma, ocular tissues from rats subjected to blast were examined for evidence of apoptosis by the detection of activated caspase 3 and TUNEL assay in their ocular tissues. METHODS: A compressed air shock tube was used to deliver 120 ± 7 KPa of BOP for duration of 2 msec to the right side of the rats. Rats were then euthanized at specific time points after blast exposure (3 hours, 24 hours, 48 hours). Ocular tissues were processed for immunohistochemistry to detect activated caspase 3 and TUNEL assay. Tissues were evaluated for relative levels of positive signal as compared to nonblast exposed controls. RESULTS: Activated caspase 3 was detected in the optic nerve, ganglion layer, and inner nuclear layer post blast exposure. At 24 and 48 hours, the inner nuclear layer from the right side had more cells with activated caspase 3. In the optic nerve, the highest levels of activated caspase 3 were detected on the right side at 24 hours post blast. CONCLUSION: BOP of 120 ± 7 KPa induces optic neuropathy and retinal damage. In both the optic nerve and retina, caspase 3 was activated in the right and left sides following blast exposure. The results of this study reveal that blast exposure induces apoptosis in both the optic nerve and retinal tissues.

Retinoid uptake, processing, and secretion in human iPS-RPE support the visual cycle.

PURPOSE: Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason, it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE. METHODS: iPS-RPE was derived from human iPS cells. Immunocytochemistry, RT-PCR, and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT), RPE65, cellular retinaldehyde-binding protein (CRALBP), and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids. RESULTS: Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol, iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media. CONCLUSIONS: iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE.

Involvement of 20alpha-hydroxysteroid dehydrogenase in the maintenance of pregnancy in mice.

The enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) catabolizes progesterone into a biologically inactive steroid, 20alpha-dihydroprogesterone (20alpha-OHP). In the corpora lutea of rats and mice, 20alpha-HSD is considered to be involved in functional luteolysis. It is also distributed in other tissues including the placenta, endometrial epithelia and fetal skin, although the roles it plays in these tissues remain to be elucidated. In the present study, we investigated the role of 20alpha-HSD in the maintenance of pregnancy using mice with targeted disruption of the 20alpha-HSD gene. We first confirmed that the number of pups was significantly smaller in 20alpha-HSD-/- pairs than in 20alpha-HSD+/+ pairs. We then mated 20alpha-HSD+/- males and females so that each pregnant female produced 20alpha-HSD+/+, 20alpha-HSD+/- and 20alpha-HSD-/- offspring. The genotype ratio of the offspring did not match the Mendel's law of inheritance, and the numbers of 20alpha-HSD+/- and 20alpha-HSD-/- offspring were smaller than expected values. Although the genotype ratio of fetuses on days 13, 15 and 18 of pregnancy matched the Mendel's law, the total number of fetuses on day 18 was significantly smaller than that on day 13, suggesting that fetal loss occurred during late pregnancy. Next, we transferred 20alpha-HSD+/+ embryos to 20alpha-HSD+/+ or 20alpha-HSD-/- females and found that the number of offspring was significantly smaller in 20alpha-HSD-/- dams than in 20alpha-HSD+/+ dams. Expression of 20alpha-HSD mRNA in the fetus, placenta and uterus progressively increased from day 11 to 18 of pregnancy. In addition, concentrations of progesterone were significantly higher in the 20alpha-HSD-/- fetuses than in the 20alpha-HSD+/+ fetuses, while those of 20alpha-OHP were lower in the 20alpha-HSD-/- fetuses than in the 20alpha-HSD+/+ fetuses. These results suggest that both maternal and fetal 20alpha-HSD play a role in maintaining normal pregnancy at least partially by reducing progesterone concentrations in fetuses.

Reproductive phenotypes in mice with targeted disruption of the 20alpha-hydroxysteroid dehydrogenase gene.

In the corpus luteum of rats and mice, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) catalyzes the conversion of progesterone to a biologically inactive metabolite, 20alpha-dihydroprogesterone (20alpha-OHP). The reduction of progesterone by 20alpha-HSD is believed to be important for functional luteolysis in these rodent species. In addition to the corpus luteum, expression of 20alpha-HSD has been demonstrated in tissues such as the placenta, endometrial epithelia, and fetal skin, although the roles it plays in the latter tissues remain to be determined. To determine the contribution of 20alpha-HSD to functional luteolysis and to the rodent reproductive system more generally, we generated a strain of mice with targeted disruption of the 20alpha-HSD gene. In the 20alpha-HSD-/- mice we obtained, which lacked the genomic region essential for catalytic reaction, neither 20alpha-HSD activity in the corpus luteum nor an increase in the serum concentrations of 20alpha-OHP during pseudopregnancy or pregnancy was detected. The durations of the estrous cycle, pseudopregnancy, and pregnancy were significantly prolonged in the 20alpha-HSD-/- mice, although the serum progesterone levels decreased to levels low enough for delivery of pups at term of pregnancy. In addition, the number of pups, especially live pups, was markedly decreased in the 20alpha-HSD-/- mice. These findings suggest that the role of 20alpha-HSD in functional luteolysis is relatively minor but that it is involved in the survival of newborn mice.